The invention relates in general to methods for detecting the presence of a selectable marker in a plant. In particular, a simple and efficient method for detecting the presence of the neomycin phosphotransferase II protein in plants is disclosed.
In the production of transgenic plants, a gene encoding a selectable marker that confers resistance to a selective agent is often included in the transformation vector to provide a means for distinguishing plant tissue that has been transformed from that which has not. The selection is typically made by growing the transformed tissue in an environment containing the selective agent and only those tissues expressing the selectable marker gene product are able to survive.
Neomycin phosphotransferase II (NPTII) is a protein of bacterial origin that confers resistance to a number of selective agents, such as kanamycin, paromomycin and genenticin, and when genetically engineered to be expressed in plant tissues, has been used as a selectable marker. Although the use of NPTII as a selectable marker to select for transformed plant tissue in the early stages of producing a transgenic plant has become routine, no efficient method for identifying NPTII-containing plants in the field has been disclosed. Typical methods involve laboratory analysis of harvested tissue from potential transgenic plants through methods including Southern blotting, immunoassays, and enzyme activity assays. These laboratory analyses are labor-intensive and time-consuming endeavors, particularly when large numbers of plants must be tested.
One method for identifying NPTII-containing transgenic tomato plants in the field has been reported. Weide et al. (Theor. Appl. Genet. 78:169-172, 1989) disclosed that young (three-leaf stage) transgenic tomato plants could be phenotypically distinguished from nontransgenic counterparts by spraying the plants with a solution containing kanamycin. In the Weide et al. process, trays of seedlings were sprayed with a kanamycin solution that did not include a surfactant over a three-day period, with a total application of 0.5-2.0 mg kanamycin per plant. Transgenic plants containing the NPTII protein were distinguished from non-transgenic plants in that transgenic plants did not develop bleached spots on the treated tissue within 7 days of treatment. NPTII activity in the putative transgenic plants was subsequently confirmed by a radiolabel transfer assay. This approach is disadvantageous in at least one respect in that it necessitated the use of large amounts of selective agent and a lengthy period of incubation before results were obtained and has not been shown to be an effective method for any plant other than tomato.
Thus, there exists a need for a rapid and more efficient method by which transgenic plants comprising a selectable marker may consistently be identified in the field.
This invention relates to an improved method for identifying plants expressing a selectable marker gene product, such as NPTII, and growing in the absence of a selective agent. More specifically, in one embodiment of the invention there is provided a method for detecting the presence of a selectable marker in a plant comprising contacting a composition comprising a selective agent and an effective amount of an organosilicone surfactant with putative transgenic plants, assessing the physical appearance of the plants for evidence of necrosis and/or bleaching of the treated plant tissue, and assigning the status of transgenic or non-transgenic to such plants based on the physical appearance of such plant tissue. A plant with little or no necrosis or bleaching evidences the presence of a selectable marker gene product in the plant and is determined to be a transgenic plant.
In a further embodiment of the present invention there is provided a method for detecting the presence of a selectable marker in a plant comprising contacting an effective amount of a selective agent with putative transgenic plant tissue and separately contacting an effective amount of an organosilicone surfactant with said putative transgenic plant tissue, assessing the physical appearance of the treated plant tissue for evidence of necrosis and/or bleaching of the treated plant tissue, and assigning the status of transgenic or non-transgenic to such plants based on the physical appearance of such plant tissue.
Among the many objects and advantages of the present invention include the provision of a method that utilizes a significantly reduced amount of the selective agent in the method as a result of the use of an organosilicone surfactant in cooperation with the selective agent; and the provision of a rapid, non-destructive method that may be utilized in field conditions on growing plants.
It has been discovered that existing methods for detecting the presence of a selectable marker gene product in a transgenic plant growing in a non-selective environment may be improved by applying an organosilicone surfactant to the plant in addition to the selective agent or by utilizing a composition comprising a selective agent with an effective amount of an organosilicone surfactant. In accordance with the invention, significantly reduced amounts of the selective agent may be used as compared to other methods. In general, subsequent to treating the putative transgenic plant tissue with the selective agent and organosilicone surfactant (whether together or separately), the physical appearance of the treated tissue is assessed to determine is whether the treated tissue contains a selectable marker gene product or not. Treated plants that contain the selectable marker gene product will exhibit one of two phenotypes:
1) no bleaching or necrosis of the treated plant tissue; or
2) reduced bleaching or necrosis relative to that seen in a similarly treated nontransgenic plant of the same genetic background. Transgenic plants are thereby distinguished from plants that have undergone the same selective agent/organosilicone treatment and do not contain the selectable marker gene product, which display relatively more bleaching and/or necrosis of the treated plant tissue.
The nucleic acid sequence serving as the source of the selectable marker gene product functions to produce a phenotype in cells that facilitates their identification relative to cells not containing the marker. Characteristics of useful selectable markers for plants, both dicotyledonous and monocotyledonous, have been outlined in a report on the use of microorganisms (Advisory Committee on Novel Foods and Processes, July 1994). These characteristics include stringent selection with minimal contaminating nontransformed tissue, high numbers of independent transformation events without interference in subsequent regenerative steps, application to a large number of species, and availability of an assay to detect the marker. Several antibiotic and herbicide resistance markers satisfy these criteria (Dekeyser et al., Plant Physiol., 90: 217-223, 1989; Della-Cioppa et al., Bio/Technology, 5:579-584, 1987). For example, NPTII confers resistance to kanamycin, paromomycin and GENENTICIN; aph IV confers resistance to hygromycin B; aac3 and aacC4 confer resistance to gentamycin; the pat and bar genes confer resistance to phosphinothricin; and the enolpyruvylshikimate-phosphate synthase (EPSPS) and glyphosate oxidoreductase (GOX) genes confer resistance to glyphosate. In a preferred embodiment of the present invention, the selectable marker confers resistance to an antibiotic and, more preferably, the selectable marker is NPTII.
The detection method of this invention may be utilized with any species compatible with transformation with a nucleic acid sequence of interest and subsequent regeneration to form a transgenic plant. The plant may be a monocotyledonous or dicotyledonous plant. More preferably, it will be monocotyledonous of the Gramineae (Poaceae) family or dicotyledonous of the Leguminosae family. Most preferably, it will be a corn (Zea mays), wheat (Triticum), cotton (Gossypium), or soybean (Glycine max) plant.
The plant may be an Acacia, alfalfa, aneth, apple, apricot, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassaya, cauliflower, celery, cherry, chicory, cilantro, citrus, clementines, coffee, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, mango, melon, mushroom, nectarine, nut, oat, oil palm, oil seed rape, okra, onion, orange, an ornamental plant, papaya, parsley, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, raspberry, rice, rye, sorghum, Southern pine, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato, triticale, turf, turnip, a vine, watermelon, yams, or zucchini.
A selective agent corresponding to the selectable marker gene product may be contacted with leaves of plants suspected of comprising the selectable marker. For example, expression of NPTII protein in plants confers resistance to a variety of selective agents, including kanamycin, paromomycin, ribostamycin, butirosin, geneticin, and combinations thereof. The selective agent may be any compound toxic to plants, wherein the selectable marker gene product reduces or eliminates the toxicity. The selective agent is preferably used in a quantity sufficient to make it possible to distinguish between plants comprising the selectable marker gene product and plants lacking the selectable marker gene product without causing indiscriminate toxicity.
In the preferred embodiment of the invention where NPTII is the selectable marker gene product, kanamycin is preferably used as the selective agent. Kanamycin is preferably applied to the plant in solution but may alternatively be applied in a solid or powdered form. In an alternate embodiment of the invention, paromomycin is preferably used as the selective agent corresponding to NPTII as the selectable marker. Paromomycin is preferably applied to the plant in solution but may alternatively be applied in a solid or powdered form. In a further embodiment of the invention, a combination of kanamycin and paromomycin is preferably used as the selective agent corresponding to NPTII as the selectable marker. Kanamycin and paromomycin are preferably applied to the plant in solution but may alternatively be applied in a solid or powdered form.
An organosilicone surfactant may further be contacted with the plant. Use of such a surfactant enhances the uptake of the selective agent, thereby reducing the total quantity of selective agent necessary for the assay and providing greater consistency of results. Any organosilicone surfactant compatible with the method of the instant invention may be used. The concentration of surfactant used may generally be any concentration suitable and effective to elicit a biological response in the treated plant when applied with a selective agent.
More preferably, a trisiloxane organosilicone surfactant may be used, and most preferably, the trisiloxane organosilicone surfactant SILWET-L77 (SILWET-L-77 is a registered trademark of OSi Specialties, Tarrytown, N.Y.) is used. SILWET L-77 is a nonionic silicone-based spray surfactant concentrate used in conjunction with a number of agricultural chemicals. SILWET L-77 is preferably used at concentrations of about 0.001% (v/v) to about 1.0% (v/v) in solution. More preferably, it is used at concentrations of about 0.01% (v/v) to about 0.08% (v/v) in solution, and most preferably, it is used at a concentration of about 0.04% (v/v) to about 0.07% (v/v) in solution.
Alternatively, other organosilicone surfactants may be used, including SILWET 408, SILWET Y-12808, SILWET L-7607, SILWET L-7602, SILWET L-7210, SILWET L-7002, SILWET L-720, and SILWET L-7200, all of which are registered trademarks of OSi Specialties, Tarrytown, N.Y. The organosilicone surfactants have the structure (CH3)3SiOxe2x80x94[(CH3)SiC3H6(CH2CH2O)y(CH2CH2CH2O)zR]xxe2x80x94OSi(CH3)3, where:
The selective agent and organosilicone solutions described above may be contacted with the plant sequentially in any order, or the selective agent may be combined in solution with the organosilicone surfactant prior to contacting them with the plant to be tested.
The concentration of surfactant and the ratio of surfactant to antibiotic may be varied depending on plant size, plant condition, humidity, temperature, and other environmental conditions. Routine experiments may be performed to optimize the effective concentrations of surfactant and antibiotic necessary for a given plant""s situation.
In particular, the stage of growth of the plant to be tested may be taken into consideration when applying the selective agent/organosilicone surfactant solutions (whether sequentially or as a combination). For example, corn development is divided into two major subdivisions; vegetative stages and reproductive stages. The vegetative stages are defined as V1 through V(n), where (n) represents the last leaf stage before tasseling occurs. The value of (n) may vary depending on hybrid and environmental differences. Each leaf stage is defined according to the uppermost leaf whose leaf collar is visible (i.e., V1=first leaf collar visible, V2=second leaf collar visible, etc.). Tested corn plants are preferably in a vegetative stage, and more preferably in V1, V2, V3, V4, V5, V6, V7, or V8. Most preferably, tested corn plants are in vegetative stage V1, V2, V3, V4, V5, or V6. Testing is done at an early stage for convenience not because the efficiency of the test is reduced. Soybean development is measured by the number of trifoliate leaves. The youngest (topmost) leaves appear to be the most sensitive to the selection agents and are therefore the preferred plant tissue to be tested in the assay.
Where the plant to be tested is a V1 or V2 corn plant, preferably about 0.0025 mg kanamycin to about 0.0125 mg kanamycin is contacted with the plant. Most preferably, about 0.01 mg kanamycin is contacted with the plant. Where the plant to be tested is a V3, V4, V5, V6, V7, or V8 corn plant, preferably about 0.005 mg kanamycin to about 0.025 mg kanamycin is contacted with the plant. Most preferably, about 0.02 mg kanamycin is contacted with the V3, V4, V5, V6, V7, or V8 corn plant. With respect to the use of paromomycin as the selective agent, where the plant to be tested is a V1 or V2 corn plant, preferably about 0.0025 mg paromomycin to about 0.0125 mg paromomycin is contacted with the plant. Most preferably, about 0.01 mg paromomycin is contacted with the V1 or V2 plant. Where the plant to be tested is a V3, V4, V5, V6, V7, or V8 corn plant, preferably about 0.005 mg paromomycin to about 0.025 mg paromomycin is contacted with the plant. Most preferably, about 0.02 mg paromomycin is contacted with the V3, V4, V5, V6, V7, or V8 plant.
When a combination of kanamycin and paromomycin is used as the selective agent and where the plant to be tested is a V1 or V2 corn plant, preferably about 0.0025 mg kanamycin to about 0.0125 mg kanamycin and 0.0025 mg paromomycin to about 0.0125 mg paromomycin are contacted with the plant. Most preferably, about 0.01 mg kanamycin and 0.01 mg paromomycin are contacted with the V1 or V2 plant. Where the plant to be tested is a V3, V4, V5, V6, V7, or V8 corn plant, preferably about 0.005 mg kanamycin to about 0.025 mg kanamycin and about 0.005 mg paromomycin to about 0.025 mg paromomycin are contacted with the plant. Most preferably, about 0.02 mg kanamycin and 0.02 mg paromomycin are contacted with the V3, V4, V5, V6, V7, or V8 corn plant.
It will be recognized that the actual concentration of the solution comprising the selective agents to be contacted with the plants in the quantities described above may be varied. Nevertheless, it is important to note that minimizing the total quantity of solution applied to the plant may improve assay sensitivity, as smaller quantities of solution are less likely to run off the plant. Therefore, the use of more concentrated solutions of selective agent is preferred. In the specific examples presented below, the selective agents are used at a concentration of 1000 mg/L in solution.
The selective agent and organosilicone surfactant may preferably be contacted with the whorl of the plant. Alternatively, they may be contacted with individual leaves, stems, or other appropriate surfaces of the plant. Most preferably, small volumes of selective agent and surfactant are contacted with the whorl of a V1, V2, V3, V4, V5, V6, V7, or V8 corn plant, from which any standing water has preferably been removed. Alternative application methods include the use of a pipetter to pipet solutions of selective agent and surfactant into the whorl, painting of leaves with solutions of selective agent and surfactant, and attachment of cotton balls soaked in solutions of selective agent and surfactant to the whorl for a period of several days. Sprays could be used, but they are environmentally less desirable. Most preferably, a pipetter will be used to pipet solutions into the whorl. For soybean and other plants without a whorl, the solution is preferably applied to the leaf.
The appearance of the treated plants is assessed following treatment. This assessment is preferably performed from three to seven days after treatment, and most preferably after five days. Treated plants lacking the selectable marker gene product display marked bleaching or necrosis, whereas plants comprising the selectable marker gene product display either reduced or no bleaching and necrosis relative to what is seen in the plants lacking the selectable marker gene product. In a preferred embodiment of the present invention where the selectable marker gene product confers resistance to an antibiotic, treatment of nontransgenic plants with a solution comprising the antibiotic may result in bleaching and/or necrosis. In particular, where the selectable marker gene product is NPTII, treatment of nontransgenic plants with kanamycin may result in bleaching, whereas treatment of nontransgenic plants with a solution comprising paromomycin may result in necrosis. The quantity of selective agent used and the quantity of selectable marker gene product in the transgenic plant may influence the appearance of the plant following treatment with surfactant and selective agent. As a consequence, appropriate selective agent and surfactant titration controls must be performed with each transgenic line of the plant to be tested and its nontransgenic counterpart.
The underlying concept and method of the instant invention may be further applied to the identification of transgenic plants that comprise other selectable markers. In these cases, a suitable quantity of the corresponding selective agent and organosilicone surfactant may be contacted with an appropriate surface of the plant by one of the methods detailed above or by other means known to those of skill in the art, and the results may be similarly assessed. A modification of the disclosed method could reasonably be expected to work for many plants that may be transformed to comprise a selectable marker.
Specific methods for transforming a wide variety of plants and obtaining transgenic plants are well documented in the literature (e.g., Gasser and Fraley, Science 244:1293, 1989; Fisk and Dandekar, Scientia Horticulturae 55: 5, 1993; Christou, Agro Food Industry Hi Tech, p.17, 1994; and the references cited therein). Any vector compatible with expression of a nucleic acid sequence comprising the selectable marker gene product in plants may be used in the generation of potentially transgenic plants to be tested by the method of the instant invention. The nucleic acid sequence may further comprise a gene of interest. The method of the instant invention may therefore be used in the indirect identification of plants comprising a gene of interest in addition to a nucleic acid sequence comprising the selectable marker gene product. Methods by which appropriate vectors may be constructed and used in the transformation of regenerable cell cultures, and by which such transformed cell cultures may be regenerated to form transgenic plants, are well known to those of skill in the art.
In a preferred embodiment of the invention, vectors may contain any NPTII-encoding sequences. NPTII proteins may include NPTII fusion proteins (for example, an NPTII/GUS fusion protein). The NPTII-encoding sequence may have altered codon usage to optimize translation in the plant. The codon usage may be modified to reflect monocotyledonous or dicotyledonous codon usage. For example, corn codon usage may be used for NPTII-encoding sequences to be used in transgenic corn.
In accordance with the present invention, one may transform any dicotyledonous or monocotyledonous plant with NPTII, with one of the other suggested selectable markers, or with any other selectable marker known to those of skill in the art, and subsequently use the method of the instant invention in the identification of successfully transformed plants.
The invention further encompasses kits to aid in the detection of transgenic plants containing a selectable marker gene product such as an antibiotic and more particularly an NPTII protein. The kits may comprise a first vessel containing an organosilicone surfactant; and a second vessel containing one or more selective agents to which the selectable marker gene product confers resistance in plants. Alternatively, the kit may comprise a vessel containing an organosilicone surfactant, and one or more selective agents to which the selectable marker gene product protein confers resistance in plants. The organosilicone surfactant may be any surfactant compatible with the selective agent, and preferably is SILWET L-77, SILWET 408, SILWET Y-12808, SILWET L-7607, SILWET L-7602, SILWET L-7210, SILWET L-7002, SILWET L-720, or SILWET L-7200.
The following definitions are provided in order to aid those skilled in the art in understanding the detailed description of the present invention.
xe2x80x9cBleachingxe2x80x9d refers to faded or absent leaf coloration, in spots or over the leaf surface.
xe2x80x9cExpressionxe2x80x9d refers to the transcription of a gene to produce the corresponding mRNA and translation of this mRNA to produce the corresponding gene product, i.e., a peptide, polypeptide, or protein.
xe2x80x9cGenexe2x80x9d refers to chromosomal DNA, plasmid DNA, cDNA, synthetic DNA, or other DNA that encodes a peptide, polypeptide, protein, or RNA molecule, and regions flanking the coding sequence involved in the regulation of its expression.
xe2x80x9cLeaf collarxe2x80x9d refers to the yellow flared band appearing at the point where the upper part of the leaf is attached to the sheath (lower part of the leaf surrounding the stem).
xe2x80x9cNecrosisxe2x80x9d refers to localized death within living tissue.
xe2x80x9cNPTIIxe2x80x9d refers to a protein capable of conferring resistance to kanamycin, paromomycin, ribostamycin, butirosin, genenticin, or a combination thereof.
xe2x80x9cSelectable markerxe2x80x9d refers to a nucleic acid sequence whose expression confers a phenotype facilitating identification of cells containing the nucleic acid sequence. Selectable markers include those that confer resistance to toxic chemicals (e.g., ampicillin resistance, kanamycin resistance), complement a nutritional deficiency (e.g., uracil, histidine, leucine), or impart a visually distinguishing characteristic (e.g., color changes or fluorescence).
xe2x80x9cSelective agentxe2x80x9d refers to a substance toxic upon exposure to cells or organisms lacking a resistance mechanism.
xe2x80x9cSILWET L-77xe2x80x9d refers to the organosilicone surfactant having the structure (CH3)3SiOxe2x80x94[(CH3)RSi]xxe2x80x94OSi(CH3)3, where x=1 and R=xe2x80x94C3H6Oxe2x80x94(C2H4O)8xe2x80x94CH3.
xe2x80x9cSurfactantxe2x80x9d refers to a surface-active agent, i.e., one which lowers surface tension at the plane of contact between phases.
xe2x80x9cTransgenexe2x80x9d refers to a nucleic acid coding sequence of interest and regions flanking the coding sequence involved in the regulation of its expression in the desired host.
xe2x80x9cTransgenicxe2x80x9d refers to organisms into which transgenes are integrated.
xe2x80x9cTrifoliatexe2x80x9d refers to a three-leaf arrangement on a soybean plant.
xe2x80x9cV1, V2xe2x80x9d etc. refer to specific stages within the vegetative period of corn development. These are defined as V1 through V(n), where (n) represents the last leaf stage before tasseling occurs and can fluctuate with environmental and hybrid differences. Each leaf stage is defined according to the uppermost leaf whose leaf collar is visible (i.e., V1=first leaf collar visible, V2=second leaf collar visible, etc.).
xe2x80x9cWhorlxe2x80x9d refers to a circular arrangement of three or more leaves, flowers, or other parts at the same point or level; here, the cluster of youngest leaves at the apex of the seedling.
The following examples are included to demonstrate preferred embodiments of the is invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed, while still obtaining a like or similar result and without departing from the spirit and scope of the invention.